Acquired immunodeficiency syndrome (AIDS), identified in 1981, was initially thought to be confined within several risk groups, different from the general population. The isolation of HIV, the infectious agent responsible for AIDS, soon followed and prompted optimistic forecasts regarding the prospects for a future vaccine. These assumptions soon proved unwarranted, since none of the conventional vaccine development strategies was efficient against HIV. At the same time, the disease spread rapidly, affecting millions of people.
Currently, there are 36.1 million AIDS cases worldwide with an estimated 5.3 million new HIV infections during the year 2000. The annual death toll is approximately 2.8 million with the majority of infected individuals in the Third World. There is currently no vaccine against HIV, and AIDS, if untreated, leads to the death of over 95% of infected individuals 10 years post-infection. The only major positive development in the field has been a recent formulation of triple-drug therapy, also called highly active antiretroviral therapy (HAART), in which patients receive a combination of different drugs targeting various viral proteins. Earlier modes of treatment did not contain the virus for long, resulting in the rapid generation of drug-resistant strains and the ultimate progression of the disease. However, while highly successful in many cases, triple-drug therapy is not without caveats.
First, it requires that patients follow the drug regimen thoroughly. Even a short discontinuation of therapy might result in virus re-emergence. Secondly, a patients' quality of life is impaired due to various drug-related side effects. And, most importantly, the cost of these necessary drugs makes them virtually unavailable to the majority of infected individuals, including those outside of health insurance nets of the industrialized world, despite recent political efforts to the contrary. Those efforts, even if they ultimately result in a dramatic slashing of drug prices, may not be able to overturn the tide of the epidemic, since in the foreseeable future, the state of public health systems in the Third World will not permit thorough AIDS diagnostics, drug distribution and monitoring of patients' adherence. Thus, an efficient HIV vaccine will present a major scientific and medical breakthrough. Even if an annual booster is required for protection, such a vaccine will be much more efficient in AIDS prevention than any drug at a reduced cost and with a lower threshold of availability to the general population. This aim of generating a safe and effective HIV vaccine has not yet been reached. In short, the generation of a vaccine against the infectious agent that attacks the immune system itself has proven to be extremely challenging. There are many reasons for this, the main of which is the ingenious way in which HIV replicates and persists in the infected host.
The enormous genetic diversity of HIV presents a major problem for vaccine development. Not only are there numerous viral phenotypes, and new ones are likely to emerge, but also the capability for permanent genetic and antigenic drift enables the virus to evade host immune response (IR) in a single infected person. It is recognized that the immune system of the infected individual mounts a vigorous anti-viral response, which contains HIV replication for a prolonged period of time. However, total viral clearance is never reached and accumulating antigenic changes finally enable the virus to avoid the action of the immune system and then, to overcome it. Similarly, HIV is likely to efficiently evade the immune response in an immunized individual if the response generated by the vaccine is too narrow or too weak.
HIV can be genetically categorized into three groups, M, O and N, with M comprising 11 different clades. While one or two clades predominate in various countries, the predominant clade changes over time due to recombination events. Thus, constructing a vaccine that is effective globally is a large undertaking, and possibly not practical. More likely it will be necessary to construct a vaccine effective against one or two clades, and even that may not be useful in the long term. Interestingly, other viruses have been classified based on serotypes, reactivity to immune sera. Classification of HIV based on immunotypes, reactivity to defined monoclonal antibodies is just beginning. Already, though, it has been demonstrated that the genotypes do not correlate with the immunotypes, and that there are fewer immunotypes than genotypes. Constructing vaccines based on immunotypes may thus be a more practical method for obtaining global efficacy for the vaccine.
There is no current theoretical consensus on the necessity of sterile immunity to protect against HIV. Proponents of this requirement point to the capacity of the virus to integrate into the DNA of infected cells and to persist in the organism. Needless to say, this feature of HIV biology provides for another major barrier against successful vaccine development. The opposing viewpoint draws on the above-mentioned capability of an organism to mount a strong anti-HIV IR and on the correlation of disease progression with viral load at the acute stage of infection (set-point). The argument is that the vaccine will already be beneficial if it diminishes viral load at the early stage of infection, thus aiding the immune system of the host. From a practical standpoint, even a partially effective HIV vaccine is thought to provide a positive impact on the AIDS epidemic and therefore sterile immunity is not a prerequisite for products currently being tested and/or developed.
HIV diversity and its capability for antigenic drift are the underlying reasons for the firmly established inefficiency of first-generation HIV vaccines, those that induced a humoral (antibody) response against a limited number of epitopes. Those vaccines used a recombinant envelope protein of HIV, gp120; considerable amount of research has been done using a closely related simian immunodeficiency virus, SIV. It has been recognized that in both cases the bulk of the IR was directed against linear epitopes, while most of the epitopes presented by actively replicating HIV are discontinuous and structure-dependent. The tertiary structure of the virus envelope was not preserved in those first-generation vaccines, having never been attained in genetically engineered gp120 that lacked proper post-translational modification. Furthermore, some of the linear epitopes exhibited high variability among different strains of HIV/SIV and a low level of antigenic cross-reactivity. Consequently, cross-protection in immunized subjects was very low.
A significant problem with an inactivated SIV vaccine was that the protection generated upon live challenge was due to the human cellular antigens present in the envelope. The SIV used as the immunogen was produced in human cells; HIV and SIV incorporate cellular antigens into their envelopes and in this case, SIV incorporated human antigens into its envelope. When macaques were immunized against the inactivated SIV preparation and then challenged with SIV grown in human cells, the macaques were protected. However, when macaques were immunized with either uninfected human cells or with purified human cellular antigens, such as MHC class II, and then challenged with SIV grown in human cells, the macaques were also protected, suggesting that the protective response was due to immunity against the cellular antigens.
Live-attenuated vaccines proved to be extremely efficient in protecting against SIV. However, immunization with live virus results in persistent infection of the vaccinee, which in the case of SIV (and likely HIV) results in an ever-occurring genetic drift and in an emergence of pathogenic viral strains from the original, defective vaccine strain in approximately 10% of animals tested. The weaker live-attenuated mutants, ones that do not replicate efficiently in the organism, seem to be cleared without inducing potent immunity. Collectively taken, this makes the use of a live-attenuated HIV vaccine impractical and unsafe, though efforts are underway to construct a severely defective HIV strain that will replicate in the organism but will not be capable of pathogenic reversion.
The work on the live-attenuated SIV model marked an important scientific breakthrough in the field of HIV vaccines. First, it was shown that protection against an HIV-like virus is possible and, second, that this protection was attained without a marked humoral response in the vaccines. That, together with the failure of inactivated and genetically engineered vaccines, directed the attention of researchers towards strategies capable of generating a vigorous cellular IR against HIV. The accumulated evidence from studying HIV-infected individuals that were able to contain the viral infection further supported this shift. Cellular immune reactions are thought to play a leading role in this containment phenomenon.
There are two main types of recombinant vaccine vectors that generate a strong cellular IR: viral and DNA-based. Of viral vectors, the most advanced in the HIV field are poxviral (using vaccinia or canarypox viruses as a backbone) or adenoviral. Other viral vectors are being actively developed as well. In these settings, recombinant HIV proteins are expressed in the vaccinee using the same transcriptional and translational machinery as the vector genes and a cellular IR results from vector persistence in the organism and its inherent immunogenicity. DNA-based vector is a plasmid containing various HIV protein genes under the control of a strong eukaryotic promoter and other features necessary for efficient transcription and translation of the recombinant gene. Direct inoculation of such a plasmid results in the generation of a cellular IR against the encoded proteins. Both DNA and viral vectors induce a substantial IR in vaccinees, but their protective effects against HIV remain to be demonstrated, although some results obtained on SIV model appear promising. There is no guarantee, however, that any of these approaches will result in a verifiable success in field studies.
The development of inactivated and recombinant protein-based vaccines has, meanwhile, entered a new phase, although there have been lingering doubts on the validity of such approach that mostly results in the generation of humoral immunity. This time, significant attention is being paid to the maintenance of the structure of virion proteins and to the inclusion of different antigenic subtypes. The recombinant protein research avenue has generated the rgp120 product (Vaxgen), which is currently in Phase III trials. The ongoing argument on the efficiency of this product may not be settled by the results of the trial since the FDA will grant a license to manufacturers should the vaccine show at least a 30% efficiency. Such a low immunization threshold standard testifies to the desperate state in which HIV vaccine research finds itself today.
In the last decade, a whole, inactivated HIV vaccine, Remune (The Immune Response Corporation, Carlsbad, Calif.), has been developed as a therapeutic vaccine and entered into clinical trials. Remune is made from a virus that contains clade A envelope and lade G gag proteins, which was inactivated by β-propiolactone and 60Co irradiation, and then formulated in incomplete Freund's adjuvant. This inactivated preparation has been shown to be safe and immunogenic. In addition to inducing a humoral response to core proteins, Remune has been shown to induce cross-clade CD4 and CD8 responses, indicating that both arms of the immune system were capable of being stimulated with an inactivated HIV vaccine. Unfortunately, possible due to the lack of gp120 on the Remune vaccine, only a poor therapeutic response was generated as indicated by modest decreases in viral load and small increases in CD4+ T cell counts in HIV-infected patients on HAART.
Recently, it was shown that a number of cysteine-modifying reagents, such as 2,2′-dithiodipyridine (aldrithiol-2; AT-2) render HIV totally non-infective by cross-linking the zinc-fingers of its core protein. This treatment does not result in any structural disruption of HIV virions. HIV and SIV preparations inactivated by this method are currently being tested for their protective capabilities. Thermal and chemical inactivation of HIV is being revisited as well with the aim to minimize irreversible conformational changes in viral proteins. Also, it has been recognized that both arms of the IR, cellular and humoral, need to be stimulated by a successful HIV vaccine and that this may be reached only by using a prime/booster combination of different reagents or vectors, similar to one of the malaria vaccines. Moreover, recent reports show that the generation of neutralizing antibodies is essential for effective natural-killer (NK) cells-directed IR against HIV. Furthermore, there are indications that a strong antiviral humoral response may abrogate AIDS disease in an experimental setting, and that the recombinant HIV immunogen may stimulate considerable cellular response on its own. Taken collectively, this bodes well for the prospects of a multi-component HIV vaccine probably consisting of a vector prime (inducing cellular IR) and inactivated virion or recombinant protein booster (generating humoral IR).
Thus, there are several questions regarding whole inactivated vaccines that remain to be answered including; (1) is protection from challenge due to viral specific immune responses capable of being generated, (2) is cross clade protection capable due to the number of viral proteins present in the whole inactivated vaccine, and (3) can increased survival times, decreased viral loads, and increased CD4+ T cell counts be generated by a therapeutic inactivated HIV vaccine? Inactivated vaccines have several benefits over subunit, live attenuated, DNA or viral vector vaccines including: (1) the immune response may be generated against multiple viral proteins, and (2) easy and inexpensive to produce. Should a whole inactivated vaccine be incorporated into the vaccine regimen, multiple methods of inactivation will need to be employed due to FDA safety requirements. Thus, additional inactivation methods will need to be explored.
The gravity of the epidemiological situation will make any efficient vaccine a highly attractive product, even if it may require an annual boost for maintenance of protection; based on contemporary scientific data, such a scenario is likely. The uses of a whole inactivated vaccine include (1) being part of a vaccine regimen using both DNA or viral vector and a whole inactivated vaccine as the boost for sterilizing immunity; (2) use of the whole inactivated vaccine for therapeutic purposes—especially in cases where HAART has failed and (3) replacement of HAART for the whole inactivated vaccine for use in third world countries that cannot afford the very expensive drugs. The necessary vaccine strategy for HIV may resemble the situation that currently exists with influenza vaccination where annual shots of an inactivated vaccine that targets the predominant viral strain, which differs from year to year, are needed. A whole-killed inactivated HIV vaccine preparation may become a valuable component for such vaccination regimen. Historically, killed HIV vaccines did not exhibit strong immunogenicity and protective efficacy for HIV infection since the thermal or chemical means of inactivation resulted in total or near-total disruption of virion structure, in particular of the denaturation of surface proteins. These concerns are addressed by a novel virus inactivation technology that employs materials known as supercritical, critical or near-critical fluids with or without polar cosolvents or entrainers and their mixtures.
As shown by illustrative example in FIG. 1, a material becomes a critical fluid at conditions that equal its critical temperature and critical pressure. A material becomes a supercritical fluid at conditions that exceed both its critical temperature and critical pressure. The parameters of critical temperature and critical pressure are intrinsic thermodynamic properties of all sufficiently stable pure compounds and mixtures. Carbon dioxide, for example, becomes a supercritical fluid at conditions that exceed its critical temperature of 31.1° C. and its critical pressure of 72.8 atm (1,070 psig). In the supercritical fluid region, normally gaseous substances such as carbon dioxide become dense phase fluids that have been observed to exhibit greatly enhanced solvating power. At a pressure of 204 atm (3,000 psig) and a temperature of 40° C., carbon dioxide has a density of approximately 0.8 g/cc, compared with a density of 0.002 g/cc at standard conditions (0° C. and 1.0 atm), and behaves much like a nonpolar organic solvent, having a dipole moment of zero debyes.
A supercritical fluid displays a wide spectrum of solvation power, as its density is strongly dependent upon temperature and pressure. Temperature changes of tens of degrees or pressure changes by tens of atmospheres can change a compound's solubility in a supercritical fluid by an order of magnitude or more. This feature allows for the fine-tuning of solvation power and the resulting fractionation of mixed solutes. The selectivity of nonpolar supercritical fluid solvents can also be enhanced by addition of compounds known as modifiers (also referred to as entrainers or cosolvents). These modifiers are typically polar organic solvents such as acetone, ethanol, methanol, methylene chloride or ethyl acetate. Varying the proportion of modifier allows wide latitude in the variation of solvent power.
In addition to their unique solubilization characteristics, supercritical fluids possess other physicochemical properties that add to their attractiveness as solvents. They can exhibit liquid-like density yet still retain gas-like properties of high diffusivity and low viscosity. The latter increases mass transfer rates, significantly reducing processing times. Additionally, the ultra-low surface tension of supercritical fluids allows facile penetration into microporous materials, increasing extraction efficiency and overall yields.
A material at conditions that border its supercritical state will have properties that are similar to those of the substance in the supercritical state. These near-critical fluids are also useful for the practice of this invention. For the purposes of this invention, a near-critical fluid is defined as a fluid which is (a) at a temperature between its critical temperature (Tc) and 75% of its critical temperature and at a pressure at least 75% of its critical pressure or (b) at a pressure between its critical pressure (Pc) and 75% of its critical pressure and at a temperature at least 75% of its critical temperature. In this definition, pressure and temperature are defined on absolute scales, e.g., Kelvin and psia, respectively. Table 1 shows how these requirements relate to some of the fluids relevant to this invention. To simplify the terminology, materials that are utilized under supercritical, near-critical conditions or exactly at their critical point with or without polar entrainers and their mixtures will be jointly referred to as “SuperFluids.”
TABLE 1Physical Properties of SuperFluidsPvap75% of Tc75% of PcFluidFormulaBP (° C.)(psia @ 25° C.)Tc(° C.)Pc(psia)(° C.)(psia)Carbon dioxideCO2−78.586031.11070−45.0803Nitrous oxideN2O−88.570036.51051−41.0788PropaneC3H8−42.113096.76164.2462EthaneC2H6−88.757032.3709−44.1531EthyleneC2H4−103.8NA9.3731−61.4548Freon 11CCl3F23.815198.163980.3480Freon 21CHCl2F8.924178.575065.6562Freon 22CHClF2−40.814096.17223.8541Freon 23CHF3−82.263026.1700−48.7525BP = Normal boiling point;Pvap = Vapor pressure;Tc = critical temperature;Pc = critical pressureSuperFluids, when compressed, exhibit enhanced solvation, penetration and expansive properties. They are utilized to penetrate and inflate viral particles. The overfilled particles are then decompressed and, as a result of rapid phase conversion, rupture at their weakest points. The aim is to introduce minimal controlled damage to the structure of the virion, rendering it non-infective. This will preserve its overall tertiary structure and, possibly, expose some internal epitopes that are usually inaccessible to the immune system. This technique is purely physical, and does not rely on denaturing heat, chemicals or irradiation.
While previous attempts at developing inactivated vaccines have led largely to disappointment, SuperFluids CFI (critical fluid inactivation) shows great promise as a technique for developing inactivated vaccines that are both safe and protective. Previous inactivated vaccines were unsuccessful due to the degradation of the surface proteins. Techniques used to inactivate HIV have included formalin treatment, detergent disruption, exposure to psoralen and ultraviolet light and treatment with β-propiolactone. Such methods are known to denature protein, chemically modify protein and nucleic acid, disrupt macromolecular interactions and otherwise decrease the ability of the inactivated vaccine to generate an effective IR. In addition, these methods often involve potentially hazardous materials; for example, β-propiolactone is considered carcinogenic. SuperFluids CFI, on the other hand, does not destroy the essential native structure of proteins and can utilize non-carcinogenic or nontoxic substances, such as carbon dioxide or nitrous oxide. Because SuperFluids CFI inactivates enveloped viruses with the potential of retaining the integrity of proteins, this technology presents great promise for the development of an effective whole inactivated vaccine against HIV. Embodiments of the present invention address these problems inherent in the prior art with the application of supercritical, critical or near-critical fluids, with or without polar cosolvents.